Effective thrombosis mediated by human prourokinase-like polypeptides with increased binding affinity for thrombosis

ABSTRACT

Human prourokinase-like polypeptides of which oligopeptides having a structure to form a covalent bond with blood clot (thrombus) through enzymatic action of human blood coagulation factor XIII are attached to the NH 2  -terminal sides of human prourokinase derivatives.

FIELD OF THE INVENTION

This invention relates to methods for the preparation of humanprourokinase-like polypeptides i.e., substantially pure humanprourokinase mutein, with binding ability through covalent bonds withblood clots, by means of gene engineering. Prourokinase mutein functionsas a thrombolytic agent.

DESCRIPTION OF RELATED ART

Human urokinase is an enzyme present in human urine in a trace amount,and can convert the inactive plasminogen to the active plasmin. Theformed plasmin can lyse blood clots. Owing to this fact, human urokinaseis clinically used widely as a thrombolytic agent.

However, human urokinase has low affinity with blood clot and is of theactive form. Therefore it activates not only plasminogen at or in thevicinity of the blood clot but also plasminogen in the circulation,resulting in formation of a large amount of plasmin in the circulation.The extensive plasmin formation in the circulation modifies platletfunction (Blood: Vol. 68, p 275, 1986) and degrades circulatingfibrinogen and clotting factors V and VIII (Blood: Vol. 68, p 1280,1986) the extensive plasmin formation also carried the risk of graveside effect of systemic hemorrhage, which often results in death(Journal of American Colleague Cardiology: Vol. 10, p 970, 1987).

The ideal thrombolytic agent should be capable of attacking thecomponents of a thrombus while sparing the circulating clotting proteinsand platlets and have an appropriate half life in vivo. In recent years,human prourokinase and tissue plasminogen activator (hereinafterreferred to as tPA) are under development as the second generationthrombolytic agents. These agents activate the plasminogen praticularllyin close proximity to a thrombus. Therefore, excessive administration ofthese thrombolytic agents carries less risk of grave side effect ofsystemic hemorrhage. However, human prourokinase has better affinitywith thrombus than human urokinase, but has not yet been shown to be ofpractical interest. The tPA has such defects as a large amount ofinhibitor (plasminogen activator inhibitor I) present in the blood, ashort half life so that therapeutic effects are not realized unless alarge amount is administered, and an occurrence of reocclusion afterartery recanalization by thrombolysis in many cases (Circulation: Vol.73, p 347, 1986; New England Journal of Medicine: Vol. 317, p 581,1987). Substances which are not inactivated irreversibly by plasminogenactivator inhibitor I and which have better affinity with blood clotthan human prourokinase are believed to be ideal thrombolytic agents.Attempts have been made to produce such substances.

A first of such substances is a chimeric substance in which the NH₂-terminal region of tPA is fused with the COOH-terminal region of humanprourokinase having an enzymatic activity to activate the plasminogen,and produced by recombinant DNA technology, with recognition that theNH₂ -terminal region of tPA has high affimity with the blood clot (TheJournal of Biological Chemistry, Vol. 268, p 10855, 1987; Thrombosis andHaemostasis, Vol. 54, p 893, 1985). However, contrary to theexpectation, this substance has less affinity to blood clot as that oftPA.

A second of the substances is a chimeric substance in which about 560amino acids at the NH₂ -terminal side which has affinity to the bloodclot is combined chemically with the COOH-terminal region of humanprourokinase, same as that described above (Biochemistry, Vol. 25, p3603, 1986). A third is a chimeric substance made by chemicallycombining an antibody against fibrin which is a major component of bloodclot, with the COOH-terminal region of human prourokinase, same as thatdescribed above (Clinical Research Abstract, Vol. 36, p 265A, 1989). Thesecond and third substances have improved affinity to blood clot;therefore, there substances would be expected to improve thethrombolytic effect. Economical manufacturing of these second and thirdsubstances is however difficult.

Further, urokinase derivatives are reported (PCT/SU84/00008:WO84/04536)and such derivatives bind with fibrin through fibrinogen chemicallylinked to urokinase beforehand. Further more, it is reported that anoligopeptide forms covalent bonds with fibrin enzymatically due to theaction of blood coagulation factor XIII and it is suggested thataffinity of urokinase with blood clot is improved by bonding sucholigopeptide with urokinase (PCT/US88/02276: WO89/00051). However theinventors of this application recognized in their study that affinity ofurokinase with blood clot is scarcely improved by bonding sucholigopeptide with urokinase.

The purpose of this invention is human prourokinase-like polypeptideswith better affinity to blood clot than that of human prourokinase, andis to provide a method to prepare the said polypeptides economically.

SUMMARY OF THE INVENTION

This invention is human prourokinase-like polypeptides (whichhereinafter may be referred to as the human prourokinase-likepolypeptides i.e., human prourokinase mutein of this invention) made byadding an oligopeptide having a structure to form a covalent bonds withblood clot through enzymatic action of the human blood coagulationfactor XIII (which hereinafter may be referred to as theoligopeptide(s)), to the NH₂ -terminal side of a human prourokinasederivative (which hereinafter may be referred to as the humanprourokinase derivative(s)).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram for the preparation of plasmid as inExample 1 and in Example 2.

FIG. 2 is a schematic diagram for the preparation of plasmid as inExample 3.

FIG. 3 is a schematic diagram for the preparation of plasmid as inReference Example 1.

FIG. 4 is a schematic diagram for the preparation of plasmid as inReference Example 2.

FIG. 5 illustrates the DNA base sequence and corresponding amino acidsequence for encoding the human prourokinase-like polypeptide describedas FCUK (-,q).

FIG. 6 illustrates the DNA base sequence and corresponding amino acidsequence for encoding the human prourokinase-like polypeptide describedas AHUK (q.q).

FIG. 7 illustrates the DNA base sequence and corresponding amino acidsequence for encoding the human prourokinase-like polypeptide describedas APUK (k,k).

FIG. 8 illustrates the DNA base sequence and corresponding amino acidsequence for encoding the human prourokinase-like polypeptide describedas APUK (k,q).

DESCRIPTION OF THE PREFERRED EMBODIMENTS

This invention is described in detail.

(A) The oligopeptides

As used in this invention, the oligopeptides are substances capable offorming covalent bonds with fibrin, a major component in the blood clot,in a human body. The inventors studied the details of the amino acidsequences of α₂.antiplasmin, fibronectin and fibrinogen γ-chain, whichare representatives of the said substances, and found that the structureof the sequence of several e.g., 10 amino acids at the NH₂ -terminalside is . . . Gln◯◯Val (or Leu)◯Pro(or Gly) . . . (where ◯ stands for anamino acid) and discovered that a peptide with such an amino acidsequence has the capability of forming covalent bonds with suchcomponents as fibrin through the enzymatic action of human bloodcoagulation factor XIII (active form). Thus this invention has beencompleted.

The oligopeptide may be any oligomer with a structure having the abilityto form covalent bonds with a blood clot through the enzymatic action ofblood coagulation factor XIII (active form). Concrete examples are thosewith the structure of . . . Gln◯◯Val (or Leu)◯Pro(or Gly) . . . Theoligopeptide is not limited to oligopeptides at the NH₂ -terminal sideof each of fibrinogen γ-chain, α₂.antiplasmin and fibronectin, as can beseen from the board object of this invention.

The following are examples of preferable oligopeptides which are usefulin the practice of this invention.

    __________________________________________________________________________    Name of the oligopeptide                                                                   Amino acid sequence                                              __________________________________________________________________________    A            (Met.)Asn.Gln.Glu.Gln.Val.Ser.Pro.Leu.Thr.Leu.Leu.Lys.           F            (Met.)Gln.Ala.Gln.Gln.Met.Val.Gln.Pro.Gln.Ser.Pro.Val.                        Ala.Val.Lys.                                                     V.sub.1      (Met.)Ala.Gln.Lys.Met.Val.Gln.Pro.Leu.Thr.Leu.Leu.Lys.           V.sub.2      (Met.)Ala.Gln.Lys.Met.Val.Gln.Pro.Gln.Thr.Leu.Leu.Lys.           V.sub.3      (Met.)Gln.Glu.Gln.Val.Ser.Pro.Gln.Thr.Leu.Leu.Lys.               V.sub.4      (Met.)Asn.Gln.Asp.Gln.Val.Ser.Pro.Gln.Thr.Leu.Leu.Lys.           V.sub.5      (Met.)Asn.Gln.Glu.Asn.Val.Ser.Pro.Gln.Thr.Leu.Leu.Lys.           V.sub.6      (Met.)Asn.Gln.ser.Met.val.ser.Pro.Gln.Thr.Leu.Leu.Lys.           __________________________________________________________________________

where (Met) is methionine which may be present in some cases.

(B) The human prourokinase derivatives

The human prourokinase derivatives include (i) those that do not containthe whole amino acid sequences of human prourokinase, (ii) thosecontaining amino acid sequences of the region necessary and sufficientto exhibit the enzymatic action for activating plasminogen (forinstance, those with defect of from the first serine to the thirdglutamic acid or from the first serine to the 143th glutamic acid of theamino acid sequence of human prourokinase) and (iii) those in which partof the amino acids is changed to a desirable form [for instance, thoseof which one or both of the 135th lysine and 156th arginine are changedto an amino acid other than basic amino acid (e.g. glutamic acid); andthose whose 157th phenylalanine is changed to an acidic amino acid (e.g.aspartic acid)].

(C) The human prourokinase-like polypeptides of this invention

The human prourokinase-like polypeptides of this invention can beproduced by combining the human prourokinase derivatives and theoligopeptides. Preferable examples are shown below in parallel to theamino acid sequence of natural human prourokinase. The amino acidsequence is illustrated only by portions different from the amino acidsequence of natural human prourokinase and the amino acid sequence intheir region by one letter abbreviation.

    __________________________________________________________________________                         amino acid sequence                                      NATURAL HUMAN PROUROKINASE                                                                         1         135      144                                                        SNELHQVP ... GKKPSSPPEELKQC                                                   156                                                                           ... RPRFKIIGG ...                                                             HUMAN PROUROKINASE-LIKE POLYPETIDES:                     AHUK(q,q)            (M)NQEQVSPLTLLKLSNELHQVP ... GQKPSSPPEELKFQC                                  ... RPQFKIIGG ...                                        APUK(q,q)            (M)NQEQVSPLTLLKLHQVP ... GQKPSSPPEELKFQC                                      ... RPQFKIIGG ...                                        APUK(k,q)            (M)NQEQVSPLTLLKLHQVP ... GKKPSSPPEELKFQC                                      ... RPQFKIIGG ...                                        APUK(k,d)            (M)NQEQVSPLTLLKLHQVP ... GKKPSSPPEELKFQC                                      ... RPRDKIIGG ...                                        APUK(k,k)            (M)NQEQVSPLTLLKLHQVP ... GKKPSSPPEELKFQC                                      ... RPRFKIIGG ...                                        FPUK(q,q)            (M)QAQQMVQPQSPVAVKLHQVP ... GQKPSSPEELKFQC                                    ... RPQFKIIGG ...                                        V.sub.1 PUK(k,q)     (M)AQKMVGPLTLLKLHQVP ... GKKPSSPPEELKFQC                                      ... RPQFKIIGG ...                                        V.sub.2 PUK(k,q)     (M)AQKMVGPQTLLKLHQVP ... GKKPSSPPEELKFQC                                      ... RPQFKIIGG ...                                        ACUK(-,q)            (M)NQEQVSPLTLLKLKFQC                                                          ... RPQFKIIGG ...                                        FCUK(-,q)            (M)QAQQMVQPQSPVAVKLKFQC                                                       ... RPQFKIIGG ...                                        __________________________________________________________________________

where (M) is methionine which may be present in some cases.

(D) Gene system of the human prourokinase-like polypeptides of thisinvention and their preparation method

The DNA segment encoding the human prourokinase-like polypeptides ofthis invention consists of the product formed by linking a DNA encodingthe amino acid sequence of oligopeptide with a structure having theability to form covalent bond with a blood clot (such as fibrin of amajor component) through the enzymatic action of human blood coagulationfactor XIII (active form) (hereinafter referred to the oligopeptide),with a DNA encoding the amino acid sequence of the human prourokinasederivative.

The DNA encoding the amino acid sequence of the oligopeptide is a DNA ofcontinuously bound codons, each of which encodes an amino acid. Allamino acid codons are applicable as the codons. Codons which are easilyexpressed in a host cell are preferably used, and also the use of codonswhich do not form a folded structure at the level of messenger RNA dueto the series of these codons is preferable. Such DNA segments aredesigned in a desirable form which can be easily prepared by chemicalsynthesis.

Examples of preferable DNA segments encoding the oligopeptides aredescribed which

    __________________________________________________________________________    Code                                                                          __________________________________________________________________________    A Amino acid sequence:                                                                    (Met)Asn.Gln.Glu.Gln.Val.Ser.Pro.Leu.Thr.Leu.Leu.Lys.                 DNA     ATG.AAC.CAG.GAA.CAG.GTG.TCT.CCG.TTG.ACT.TTG.CTT.AAG.              F Amino acid sequence:                                                                    (Met)Gln.Ala.Gln.Gln.Met.Val.Gln.Pro.Gln.Ser.Pro.Val.                 DNA     ATG.CAG.GCA.CAA.CAG.ATG.GTT.CAA.CCT.CAG.TCA.CCG.GTT.                          Ala.Val.Lys.                                                                  GCT.GTT.AAG.                                                      V.sub.1 Amino acid sequence:                                                              (Met)Ala.Gln.Lys.Met.Val.Gln.Pro.Leu.Thr.Leu.Leu.Lys.                 DNA     ATG.GCA.CAA.AAA.ATG.GTT.CAG.COG.CTG.ACC.TTG.CTT.AAG.              V.sub.2 Amino acid sequence:                                                              (Met)Ala.Gln.LyMet.Val.Gln.Pro.G1n.Thr.Leu.Leu.Lys.                   DNA     ATG.GCA.CAA.AAA.ATG.GTT.CAG.CCG.CAA.ACT.CTT.CTT.AAG.              V.sub.3 Amino acid sequence:                                                              (Met).Gln.Glu.Gln.Val.Ser.Pro.                                        DNA     ATG CAG GAA CAG GTG TCT CCG                                                   Gln.Thr.Leu.Leu.Lys.                                                          CAG ACT TTG CTT AAG                                               V.sub.4 Amino acid sequence:                                                              (Met).Asn.Gln.Asp.Gln.Val.Ser.                                        DNA     ATG AAC CAG GACCAG GTG TCT                                                    Pro.Gln.Thr.Leu.Leu.Lys.                                                      CCG CAG ACT TTG CTT AAG                                           V.sub.5 Amino acid sequence:                                                              (Met).Asn.Gln.Glu.Asn.Val.Ser.                                        DNA     ATG AAC CAG GAA AAC GTG TCT                                                   Pro.Gln.Thr.Leu.Leu.Lys.                                                      CCG CAG ACT TTG CTT AAG                                           V.sub.6 Amino acid sequence:                                                              (Met).Asn.Gln.Ser.Met.Val.Ser.                                        DNA     ATG AAC CAA TCT ATGGTG TCT                                                    Pro.Gln.Thr.Leu.Leu.Lys.                                                      CCG CAG ACT TTG CTT AAG                                           __________________________________________________________________________

where (Met) is methionine which may be present in some cases.

A DNA segment encoding the human prourokinase derivative is obtainedfrom DNA encoding natural human prourokinase. In this case, morepreferable results are obtained if a DNA encoding natural humanprourokinase is changed to a codon which is easily expressed in the hostcell used or to a codon which does not form a folded structure at thelevel of messenger RNA, though the DNA can be used as it is.

A human prourokinase-like polypeptide of this invention from which thefirst serine to the third glutamic acid are deleted is accumulated in E.coli cells in a larger amount than human prourokinase with no aminoacids deleted.

Examples of the preferred human prourokinase derivatives are described,as follows.

(1) Derivatives in which the codon encoding the second to eighth aminoacids from the NH₂ -terminal side of the amino acid sequence of naturalhuman prourokinase is a codon described in 1 below (in the case of humanprourokinase with defect of the first to third amino acids, the codonencoding the amino acids of the fourth Leu to the eighth Pro): ##STR1##

(2) In the case of a human prourokinase derivatives with defect from thefirst serine to 143th glutamic acid, derivatives in which the codonencoding leucine and lysine corresponding to the 144th and 145th is acodon described in 2 below: ##STR2##

(3) In the case of a mutein in human prourokinase derivatives whose156th arginine is changed to glutamine, derivatives of which the codonencoding the amino acids form the 156th glutamine to 162th glycine is acodon described in 3, below ##STR3##

The DNA fragment encoding the human prourokinase-like polypeptides ofthis invention can be inserted to the expression plasmid. Concrete DNAbase sequence and corresponding amino acid sequence are shown in FIG. 5(FCUK(-,q)), FIG. 6 (AHUK(q,q)), FIG. 7 (APUK(k,k)) and FIG. 8(APUK(k,q)). The intended product is accumulated in the host cell or inthe culture solution by culturing the transformant (host cell)transformed by the transduction of the expression plasmid. When E. coliis used as a host cell, the intended product is typically obtained as aninsoluble precipitate after cell disruption by such means asultrasonication or Gaulin homogenizer.

The precipitate is dissolved in an aqueous solution such as of guanidinehydrochloride or urea, and then oxidized by air in the presence of athiol compound in order to reconstruct the original steric structure ofthe intended product. It is then purified, for example by fractionalprecipitation by salting out with ammonium sulfate, hydrophobicchromatography, or metal chelate chromatography. Generally employedbiochemical purification technologies other than the above can beapplied.

The human pruorokinase-like polypeptides of this invention are thusprepared with economical advantage.

This invention is further described in detail by reference to thefollowing examples. The operative conditions including restrictionenzymes, isolation methods of DNA fragments and synthesized doublestrand DNAs used in the examples are described below:

(1) Reaction of each restriction enzyme

Into 50 μl of each of the following reaction solutions containing 1 μgof DNA (plasmid or DNA fragment) was added 10 units of each restrictionenzyme, and the solutions were incubated at a relevant temperaturedescribed below for two hours. When partial digestion was carried out, 1to 2 units of restriction enzyme was added and the solutions wereincubated for 0.5 to an hour.

    __________________________________________________________________________    Composition of reaction solution (m mole)                                                            mercapto-                                                                           bovine                                           restriction            ethanol                                                                             serum                                                                              reaction                                    enzyme                                                                              Tris-hydrochloric                                                                      NaCl    (dithio                                                                             albumin                                                                            temperature                                 name  acid buffer (pH)                                                                       (KCl)                                                                             MgCl.sub.2                                                                        threitol)                                                                           (%)  (°C.)                                __________________________________________________________________________    Aat II                                                                              10  (7.5)                                                                              (50)                                                                              10  1     --   37                                          Acc III                                                                             10  (8.5)                                                                              200 7   7     --   60                                          Axy I 10  (7.5)                                                                              100 7   7     --   37                                          Afl II                                                                              10  (8.0)                                                                              (40)                                                                              7   7     0.01 37                                          Bal I 20  (8.5)                                                                              --  7   7     0.01 37                                          BamH I                                                                               6  (7.9)                                                                              150 6   --    --   30                                          Ban II                                                                               6  (7.4)                                                                              50  6   10    --   37                                          BspH I                                                                              10  (7.4)                                                                              (100)                                                                             10  --    0.01 37                                          Dra II                                                                              10  (8.0)                                                                              (40)                                                                              7   7     --   37                                          EcORI 100 (7.5)                                                                              50  5   --    --   37                                          Eco47 III                                                                           10  (8.5)                                                                              (80)                                                                              7   7     0.01 37                                          Hind III                                                                            10  (7.5)                                                                              60  7   --    --   37                                          Kpn I 10  (7.5)                                                                              --  7   7     --   37                                          Nar I   6 (7.4)                                                                              --  6   6     --   37                                          Nae I 10  (8.0)                                                                              20  1   6     0.01 37                                          Nco I 10  (8.5)                                                                              80  7   --    0.01 37                                          Pst I 10  (7.5)                                                                              100 10  --    --   37                                          Sca I  6  (7.4)                                                                              100 6   (1)   --   37                                          __________________________________________________________________________

(2) Blunting reaction of DNA with T₄ DNA polymerase

Into 50 μl of the following reaction solution containing 1 to 2 μg oflinear DNA was added 0.5 to 1 unit of T₄ DNA polymerase to incubate at37° C. for an hour.

Composition of reaction solution:

67 mM of Tris hydrochloric acid (pH 8.8), 6.7 mM of MgCl₂, 16.6 μM of(NH₄)₂ SO₄, 10 mM of β-mercaptoethanol, 6.7 μM ofethylenediaminetetraacetic acid, 0.0167% of bovine serum albumin, 330 μMof dCTP, 330 μM of daTP, 330 μM of dGTP and 330 μM of dTTP.

(3) Blunting reaction of DNA with Klenow fragment

Into 50 μl of the following reaction solution containing 1 to 2 μg oflinear DNA was added 0.5 to 1 unit of Klenow fragment to incubate at 25°C. for an hour.

Composition of reaction solution:

67 mM of potassium phosphate buffer (pH 7.4), 6.7 mM of MgCl₂, 1 mM ofβ-mercaptoethanol, 33 μM of dATP, 33uμM of dTTP, 33 μM of dGTP and 33 μMof dCTP.

(4) Ligation reaction of DNA by T₄ DNA ligase

Into 7.5 μl of DNA solution containing (about 0.1 μg of) DNA fragment tobe ligated were mixed 60 μl of solution A of "DNA ligation kit" made byTakara Shuzo Co. and 7.5 μl of Solution B of the same (containing T₄ DNAligase) to incubate at 16° C. for 30 minutes.

(5) Isolation of DNA fragment and others

After each plasmid was digested with restriction enzyme, the intendedDNA fragment was isolated by agarose electrophoresis. The DNA fragmentobtained by electric elution was extracted with phenol and chloroform,and purified by precipitation with ethanol. E. coli JM103 strain wastransformed by each plasmid prepared. The transformant was investigatedby quick isolation method by alkaline lysis, and a clone with theintended plasmid was obtained.

(6) Synthesized double helical DNA

The synthesized double strand DNAs used to prepare an intended plasmidwere produced according to the following procedure: Each single chainDNA oligomer was synthesized by amidite method, purified by OPCcartridge (Applied Biosystem Co.), and dried. Each pair of isolated DNAwas dissolved in a buffer (pH 7.6) of 20 mM of Tris-hydrochloric acidand 10 mM of MgCl₂, heated at 95° C. for 2 minutes, gradually cooleddown to room temperature, and then kept at 12° C. for over night toanneal. The double strand DNAs described below were thus obtained:

    __________________________________________________________________________    Name of the Synthesize double helical DNA                                     __________________________________________________________________________    HL-A  5'-TTAAGCTGAGCAACGAGCT-3'                                               HL-B  3'-CGACTCGTTGC-5═                                                   CUK-A 5'-CAAGTTTCAGTGTGG-3'                                                   CUK-B 3'-TCGAGTTCAAAGTCACACC-5'                                               AP-A  5'-CATGAACCAGGAACAGGTGTCTCCGTTGACTTTGCTTAAGCT-3'                        AP-B  3'-TGCAGTACTTGGTCCTTGTCCACAGAGGCAACTGAAACGAAT-5'                        FN-A  5'-CATGCAGGCACAACAGATGGTTCAACCTCAGTCACCGGTTGCTG-3'                      FN-B  3'-GTCCGTGTTGTCTACCAAGTTGGAGTCAGTGGCCAACGACAATT-5'                      V.sub.1 -A                                                                          5'-CATGGCACAAAAAATGGTTCAGCCGCTGACCTTGC-3'                               V.sub.1 -B                                                                          3'-CGTGTTTTTTACCAAGTCGGCGACTGGAACGAATT-5'                               V.sub.2 -A                                                                          5'-CATGGCACAAAAAATGGTTCAGCCGCAAACTCTTC-3'                               V.sub.2 -B                                                                          3'-CGTGTTTTTTACCAAGTCGGCGTTTGAGAAGAATT-5'                               V.sub.3 -A                                                                          5'-CATGCAGGAACAGGTGTCTCCGCAGACTTTGCTTAAGCT-3'                           V.sub.3 -B                                                                          3'-TGCAGTACGTCCTTGTCCACAGAGGCGTCTGAAACGAAT-5'                           V.sub.4 -A                                                                          5'-CATGAACCAGGACCAGGTGTCTCCGCAGACTTTGCTTAAGCT-3'                        V.sub.4 -B                                                                          3'-TGCAGTACTTGGTCCTGGTCCACAGAGGCGTCTGAAACGAAT-5'                        V.sub.5 -A                                                                          5'-CATGAACCAGGAAAACGTGTCTCCGCAGACTTTGCTTAAGCT-3'                        V.sub.5 -B                                                                          3'-TGCAGTACTTGGTCCTTTTGCACAGAGGCGTCTGAAACGAAT-5'                        V.sub.6 -A                                                                          5'-CATGAACCAATCTATGGTGTCTCCGCAGACTTTGCTTAAGCT-3'                        V.sub.6 -B                                                                          3'-TGCAGTACTTGGTTAGATACCACAGAGGCGTCTGAAACGAAT-5'                        TUK1-A                                                                              5'-GCCTCAGTTTAAAATCATTGGCGGCG-3'                                        TUK1-B                                                                              3'-AGTCAAATTTTAGTAACCGCCGCTTA-5'                                        TUK2-A                                                                              5'-GCCTCGCGATAAAATCATTGGCGGCG-3'                                        TUK2-B                                                                              3'-AGCGCTATTTTAGTAACCGCCGCTTAA-5'                                       CROSD-A                                                                             5'-CTAGATAAGGAGGTGAAAACCATG-3'                                          CROSD-B                                                                             3'-TATTCCTCCACTTTTGGTAC-5'                                              __________________________________________________________________________

EXAMPLE 1 Preparation of plasmid pACUK (-,q)/5, plasmid pFCUK (-,q)/5,plasmid pV₁ PUK (k,q)/5 and plasmid pV₂ PUK (k,q)/5

About 3.9 kb of DNA fragment isolated after plasmid pHUK (q,q)/0 wasdigested with restriction enzymes Bal I and Sac I and synthesizedoligomer CUK-A/B were linked by T₄ DNA ligase to give plasmid pCUK(-,q)/0.

About 3.9 kb of DNA fragment isolated after plasmid pCUK (-,q)/0 wasdigested with restriction enzymes Aat II and Sac I and synthesizedoligomer AP-A/B were linked by T₄ DNA ligase to give plasmid pACUK (-,q)/0 .

Plasmid p9420 was digested with restriction enzyme Nco I and treatedwith alkaline phosphatase and a DNA fragment containing urokinase genewhich was isolated after plasmid pACUK (-,q)/0 was digested withrestriction enzyme BspH I were linked by T₄ DNA ligase to give plasmidpACUK (-,q)/5.

About 5.4 kb of DNA fragment isolated after plasmid pACUK (-,q)/5 wasdigested with restriction enzymes Aat II and Afl II, about 0.5 kb of DNAfragment isolated after plasmid p9410 was digested with restrictionenzymes Aat II and Nco I, and synthesized oligomer FN-A/B were linked byT₄ DNA ligase to give plasmid pFCUK (-,q)/5.

About 5.8 kb of DNA fragment isolated after plasmid pAPUK (k,q)/5 wasdigested with restriction enzymes Aat II and Afl II, about 0.5 kb of DNAfragment isolated after plasmid p9410 was digested with restrictionenzymes Aat II and Nco I, and synthesized oligomer V₁ -A/B were linkedby T₄ DNA ligase to give plasmid pV₁ PUK (k,q)/5.

About 5.8 kb of DNA fragment isolated after plasmid pAPUK (k,q)/5 wasdigested with restriction enzymes Aat II and Afl II, about 0.5 kb of DNAfragment isolated after plasmid p9410 was digested with restrictionenzymes Aat II and Nco I, and synthesized oligomer V₂ -A/B were linkedby T₄ DNA ligase to give plasmid PV₂ PUK (k, g/5.

EXAMPLE 2 Preparation of plasmid pAPUK (q,q)/5 and plasmid pFPUK (q,q)/5

About 5.3 kb of DNA fragment isolated after plasmid pHUK (q,q)/0 wasdigested with restriction enzymes Aat II and Sac I and synthesizedoligomer AP-A/B were linked by T₄ DNA ligase to give plasmid pAPUK(q,q)/0.

About 1.2 kb of DNA fragment isolated after plasmid pAPUK (q,q)/0 wasdigested with restriction enzymes Afl II and BamH I and about 5.1 kb ofDNA fragment isolated after plasmid pACUK (-,q)/5 was digested withrestriction enzymes Afl II and BamH I were linked by T₄ DNA ligase togive plasmid pAPUK (q,q)/5.

About 5.8 kb of DNA fragment isolated after plasmid pAPUK (q,q)/5 wasdigested with restriction enzymes Aat II and Afl II, about 0.5 kb of DNAfragment isolated after plasmid p9410 was digested with restrictionenzymes Aat II and Nco I and synthesized oligomer FN-A/B were linked byT₄ DNA ligase to give plasmid pFPUK (q,q)/5.

EXAMPLE 3 Preparation of plasmid pAHUK (q,q)/5, plasmid pAPUK (k,k)/5,plasmid pAPUK (k,q)/5 and plasmid pAPUK (k,d)/5

About 4.9 kb of DNA fragment isolated after plasmid pAPUK (q,q)/5 wasdigested with restriction enzymes Afl II and BamH I, about 1.4 kb of DNAfragment isolated after plasmid pHUK (q,q)/0 was digested withrestriction enzymes BamH I and Sac I and synthesized oligomer HL-A/Bwere linked by T₄ DNA ligase to give plasmid pAHUK (q,q)/5.

E. coli HB101 containing plasmid pAHUK (q,q)/5 is deposited as FERMBP-2467 to the Fermentation Research Institute, Agency of IndustrialScience & Technology, Ministry of International Trade and Industry.

About 5.2 kb of DNA fragment isolated after plasmid pAPUK (q,q)/5 wasdigested with restriction enzymes Hind III and Pst I and about 1.1 kb ofDNA fragment isolated after plasmid pMUT4H was digested with restrictionenzymes Hind III and Pst I were linked by T₄ DNA ligase to give plasmidpAPUK (k,k)/5.

E. coli HB101 containing plasmid pAPUK (k,k)/5 is deposited as FERMBP-2465 to the Fermentation Research Institute, Agency of IndustrialScience & Technology, Ministry of International Trade and Industry.

About 5.5 kb of DNA fragment isolated after plasmid pAPUK (k,k)/5 wasdigested with restriction enzymes Bal I and BamH I and about 0.75 kb ofDNA fragment isolated after plasmid pCUK (-,q)/0 was digested withrestriction enzymes Bal I and BamH I were linked by T₄ DNA ligase togive plasmid pAPUK (k,q)/5.

E. coli HB101 containing plasmid pAPUK (k,q)/5 is deposited as FERMBP-2466 to the Fermentation Research Institute, Agency of IndustrialScience & Technology, Ministry of International Trade and Industry.

About 5.5 kb of DNA fragment isolated after plasmid pAPUK (k,k)/5 wasdigested with restriction enzymes Bal I and BamH I and about 0.75 kb ofDNA fragment isolated after plasmid pHUK (q,d)/0 was digested withrestriction enzymes Bal I and BamH I were linked by T₄ DNA ligase togive plasmid pAPUK (k,d)/5.

EXAMPLE 4 Expression of human prourokinase-like polypeptides gene by E.coli

Plasmids pAHUK (q,q)/5, pAPUK (q,q)/5, pAPUK (k,q)/5, pAPUK (k,d)/5,pAPUK (k,k)/5, pFPUK (q,q)/5, pACUK (-,q)/5, pFCUK (-,q)/5, pV₁ PUK(k,q)/5 and pV₂ PUK (k,q)/5, which were obtained in Examples 1, 2 and 3,were each transformed to E. coli JM103 strain according to a usualmethod. Each of the obtained transformants was cultured aerobically in50-ml of L-broth at 37° C. When the absorbance at 600 nm became about1.0, 0.5 ml of 100 mM isopropyl thiogalactopyranoside (IPTG) was added,and culture was continued for another 4 hours to express each humanprourokinase-like polypeptide gene.

EXAMPLE 5 Extraction and purification of gene products from E. coli

10 g of each of wet cells obtained in Example 4 was suspended in 100 mlof 10 mM glycine-NaOH buffer (pH 9.0, and containing 0.1M of NaCl and 5mM of EDTA), to which 50 mg of lysozyme was added and stirred for anhour while cooled with ice. Then the cells were disrupted byultrasonication. Immediately after the disruption, the cells werecentrifuged at 4° C. and 12,000 rpm for 30 minutes to give precipitate.The obtained precipitate was washed with 10 mM glycine-NaOH buffer (pH9.0) several times, and dissolved with stirring in a solution of 500 mlof 0.1M glycine-NaOH buffer (pH 9.0) to which 500 ml of 8M guanidinehydrochloride was added. Deionized water, 1M glycine-NaOH buffer (pH9.0), EDTA and reduced glutathione were added to the resulting solutionto make 4 L (final concentration: 1.0M of guanidine hydrochloride, 0.04Mof glycine, 1mM of EDTA and 0.2 mM of reduced glutathione). The obtainedsolution was let stand at room temperature over night for refolding. Thesolution was concentrated by ultrafiltration (exclusion molecularweight: 6000). A crude product was obtained from the solution by saltingout with 25% to 55% saturation of ammonium sulfate. The crude productwas dissolved in 50 mM phosphate buffer (pH 7.5, and containing 0.5M ofguanidine hydrochloride and 0.6M of ammonium sulfate), and insolublematter was removed by centrifugation. The supernatant was absorbed in aphenyl Sepharose (Pharmacia Co.) column which was equilibrated with thesame buffer, and sufficiently washed with the same buffer, then elutedwith the same buffer but not containing ammonium sulfate to collect theelution fraction containing the intended product. This fraction wasadsorbed on a Zn-chelate (Cu chelate for ACUK (-,q) and FCUK (-,q))Sepharose (Pharmacia Co.) column, sufficiently washed with 50 mMphosphate buffer (pH 6.0, and containing 0.5M of NaCl), and eluted with20 mM acetic acid buffer (pH 5.4, and containing 5M of NaCl). Theelution fraction containing the intended product was concentrated anddesalted using a ultrafiltration membrane PM-10 (Amicon Co.) (ifnecessary, it was passed through a benzamidine Sepharose column toremove the active form).

Thus a purified product corresponding to each of the humanprourokinase-like polypeptides of this invention, AHUK (q,q), APUK(q,q), APUK (k,q), APUK (k,d), APUK (k,k), FPUK (q,q), ACUK (-,q), FCUK(-,q), V₁ PUK (k,q) and V₂ PUK (k,q), was obtained.

REFERENCE EXAMPLE 1 Preparation of plasmids pMUT90pml and p9420

A DNA fragment containing prelilac p/o, which was isolated after plasmidpPLL was digested with restriction enzymes Aat II and Xba I, and about3.4 kb of DNA fragment containing laqI^(q) gene, which was isolatedafter plasmid pMJR1560 (Amersham Co.) was digested with restrictionenzyme Pst I, followed by treatment with T₄ DNA polymerase and digestionwith restriction enzyme Aat II, and synthesized oligomer CroS/D-A/B werelinked with T₄ DNA ligase to give plasmid p9410.

DNA fragment containing vector fragment, which was isolated after pMUT8Lharboring natural human prourokinase gene was digested with restrictionenzyme Nco I, was linked by T₄ DNA ligase to give plasmid p6514.

Plasmid p6514 was digested with restriction enzyme EcoR I and followedby treatment with Klenow fragment to fill in the restriction site, thenlinked by T₄ DNA ligase to give plasmid p6516.

About 4.2 kb of DNA fragment isolated after plasmid p6516 was digestedwith restriction enzyme BamH I was treated with Klenow fragment to fillin the restriction site, then linked by T₄ DNA ligase to give plasmidp6610.

About 3.7 kb of DNA fragment isolated after plasmid pMUT9Lpml (with ahuman prourokinase derivatives gene of which lysine at the 135thposition of the amino acid sequence of natural human prourokinase wassubstituted by glutamine), which was produced from plasmid pMUT8L, wasdigested with restriction enzymes Nar I and Nco I, and about 0.6 kb ofDNA fragment isolated after plasmid p6610 was digested with restrictionenzymes Nar I and Nco I were linked by T₄ DNA ligase to give plasmidpMUT90pml.

A DNA fragment containing prelilac p/o, which was isolated after plasmidp9410 was digested with restriction enzyme Kpn I, treated with T₄ DNApolymerase and further digested with restriction enzyme Aat II, andabout 3.5 kb of DNA fragment containing tac p/o, which was isolatedafter plasmid pMUT90pml was digested with restriction enzyme Pst I,treated with T₄ DNA polymerase and further digested with restrictionenzyme Aat II were linked by T₄ DNA ligase to give plasmid p9420.

REFERENCE EXAMPLE 2 Preparation of plasmids pHUK (q,d)/0 and pHUK(q,q)/0

About 4.4 kb of DNA fragment isolated after plasmid pMUT8L harboringhuman prourokinase gene was digested with restriction enzymes Axy I andHind III was treated with Klenow fragment to fill in the restrictionsite, then linked by T₄ DNA ligase to give plasmid pMUT4H.

About 3.1 kb of DNA fragment isolated after plasmid pMUT4H was digestedwith restriction enzymes Acc III and Nar I and about 1.3 kb of DNAfragment isolated after plasmid pMUT90pml was digested with restrictionenzymes Acc III and Nar I were linked by T₄ DNA ligase to give plasmidp6028.

About 3.7 kb of DNA fragment isolated after plasmid p6028 was digestedwith restriction enzymes Eco47 III and Hind III was treated with Klenowfragment to fill in the restriction site, then linked by T₄ DNA ligaseto give plasmid p6N17.

About 3.7 kb of DNA fragment isolated after plasmid p6N17 was digestedwith restriction enzymes Dra II and EcoR I, and each of synthesizedoligomers TUK1-A/B and TUK2-A/B were linked by T₄ DNA ligase to giveplasmid p7214 and plasmid p7224, respectively.

About 3.8 kb of DNA fragment isolated after plasmid pMUT90pml wasdigested with restriction enzyme Ban II, and each of about 0.5 kb of DNAfragments isolated after plasmids p7214 and p7224 were each digestedwith restriction enzyme Ban II were linked by T₄ DNA ligase to giveplasmids pHUK (q,q)/0 and pHUK (q,d)/0, respectively.

REFERENCE EXAMPLE 3 Preparation of urokinase-like polypeptide ATUK (q,q)of which a peptide with ability to form bond with fibrin is added to theNH₂ terminal side

100,000 units of purified sample of human prourokinase-like polypeptideAHUK (q,q) obtained in Example 5 was dissolved in 100 ul of 50 mM Trishydrochloric acid buffer (pH 7.5) containing 0.1M NaCl, to which 0.015cu of serum plasmin (Green Cross Co.) was added to incubate at 37° C.for an hour. Then 10 μg of trypsin inhibitor (Sigma Chemical Co.) wasadded to stop the reaction. The product was adsorbed in benzamidineSepharose column, and eluted with 20 mM acetic acid buffer (pH 4.5)containing 0.5M NaCl. The eluted fractions were concentrated anddesalted using ultrafiltration membrane PM-10 (AMICON Co.) to giveurokinase-like polypeptide ATUK (q,q). Analysis of the amino acidsequence in the neighborhood of the NH₂ terminal confirmed that thepurified sample was urokinase-like polypeptide with the same sequence asthat in the neighborhood of the NH₂ terminal of prourokinase-likepolypeptide AHUK (q,q).

It is believed that the human prourokinase-like polypeptides of thisinvention bind by covalent bonding with blood clots through theenzymatic action of the blood coagulation factor XIII (active form)which appears at the region where blood clots are formed, and then areconverted to activated form which does not leave from the blood clot.Therefore the polypeptides of this invention can be an idealthrombolytic agent which is very clot-specific, has less side effectssuch as systemic hemorrhage, and is not inhibited by plasminogenactivator inhibitor I. The polypeptides of this invention can beeconomically manufactured by gene engineering methods.

TEST EXAMPLE 1 Fibrin binding ability test

The binding ability of each of purified human prourokinase-likepolypeptides of this invention (hereinafter referred to as test sample)to a fibrin clot was compared with that of tPA, human prourokinase andurokinase-like polypeptide ATUK (q,q). The binding test was carried outaccording to the following two methods:

Reagents:

1) Tris-hydrochloric acid buffer; 50 mM of Tris, 38 mM of NaCl, 0.01% ofTriton X-100, pH 7.5

2) Fibrinogen solution; 10 mg of plasminogen free human fibrinogen ofSigma Chemical Co./ml of Tris-hydrochloric acid buffer

3) Thrombin solution; 100 units of human thrombin of Sigma ChemicalCo./ml of Tris-hydrochloric acid buffer

4) Plasmin solution; 15 cu of serum plasmin of Green Cross Co./ml of H₂O

5) Trypsin inhibitor solution; 5 mg of trypsin inhibitor of SigmaChemical Co./ml of H₂ O

6) Hirudin; Hirudin of Sigma Chemical Co.

7) Test sample solution; 5000 international units of test sample/ml ofTris-hydrochloric acid buffer

I. Testing method of binding ability in fibrin clotting process

Into a 1.5-ml eppendorf tube, while cooled with ice, were placed 75 μlof fibrinogen solution, 5 μl of 0.1M CaCl₂ aqueous solution and 100 μofeach test sample, to which the Tris-hydrochloric acid buffer was addedto make 297 μl 1 and mixed well. 3 μl of thrombin solution was added tothe solution to mix to treated for 60 minutes in a water bath of 37° C.Fibrin clots were thus formed. Immediately the clots were centrifuged at4° C. (16,000 rpm, 10 minutes). The precipitate was washed withTris-hydrochloric acid buffer. 1 NIH of hirudin and 5 to 15 μl ofplasmin solution were added to dissolve the precipitate andsimultaneously the test sample was activated. A trypsin inhibitor ofvolume of 5 fold-weight of plasmin was added to inhibit the plasminactivity. An amount of test sample was measured from the degradationactivity of synthesized substrate S-2444 of Kabi Co., being the amountof test sample bound.

II. Testing method of binding ability after fibrin clots are formed

Method I was repeated except that fibrin clots were formed with noaddition of test sample, then 100 μl of test sample solution and 200 μlof Tris-hydrochloric acid buffer were added and maintained for 60minutes in a water bath of 37° C. An amount of test sample in thecentrifuged precipitate obtained by the same treatment as that in I wasmeasured.

In the above testing method, if the test samples were human prourokinaseand APUK (k,k), the test was carried out in the presence of hirudin inorder to prevent the test sample from losing activity by degradation bythrombin.

The binding ability of each of test samples to fibrin clots are shown inthe following table:

    ______________________________________                                         ##STR4##                                                                                               binding ratio (%) after                                        binding ratio (%) in                                                                         fibrin clotting were                                test samples                                                                             fibrin clotting process                                                                      formed                                              ______________________________________                                        tPA        66             51                                                  human urokinase*                                                                          6              4                                                  HUK*       10              7                                                  HUK (q,q)   7              6                                                  ATUK (q,q) 11              7                                                  AHUK (q,q) 59             48                                                  APUK (q,q) 58             45                                                  APUK (k,q) 43             32                                                  APUK (k,d) 64             55                                                  APUK (k,k) 52             45                                                  ACUK (--,q)                                                                              31             22                                                  FPUK (q,q) 62             28                                                  FCUK (--,q)                                                                              35             21                                                  V.sub.1 PUK (k,q)                                                                        52             30                                                  V.sub.2 PUK (k,q)                                                                        48             32                                                  ______________________________________                                         *human urokinase: urokinase extracted from human urine                        *HUK: human prourokinase                                                 

The tPA and APUK (q,q), which were bound in the fibrin clotting process,left from the fibrin clots after bound were estimated at an hourlyinterval. It was recognized that tPA was left 23% after 2 hours andgradually was being left after it. To contrast, 8% of APUK (q,q) wasleft after 2 hours and no leaving was recognized after it.

TEST EXAMPLE 2 Fibrin lysis activity test

The lysis activity of each purified human prourokinase-like polypeptidesof this invention (hereinafter referred to as test sample) to fibrin wasmeasured using the standard urokinase (distributed by the NationalInstitute of Hygiene Science) as a reference.

Testing method

Into 5 ml of 0.1M phosphate buffer (pH 7.4) containing 2% of bovinefibrinogen (Seikagaku Kogyo Co.) and 10 mM of CaCl₂ were added 5 ml of0.25% aqueous solution of agarose which was heated to dissolve and 0.1ml of bovine thrombin (Mochida Pharmaceutical Co.) solution (100 activeunits/ml). After mixing, the mixture was poured in a Petri dish of 9.0cm in diameter to make a fibrin plate. On the fibrin plate was spottedeach of 5 μl of test samples, each of which contained one unit ofhirudin, and incubated at 37° C. for 18 hours. An activity value wascalculated from the lysis circle. The lysis activity of each test sampleper unit absorbance at 280 nm is shown in the following table ininternational unit.

    ______________________________________                                                      Fibrin lysis activity                                           Test sample name                                                                            International unit/O.D.280                                      ______________________________________                                        AHUK (q,q)     8.8 × 10.sup.4                                           APUK (q,q)    10.9 × 10.sup.4                                           APUK (k,q)    10.4 × 10.sup.4                                           APUK (k,d)     8.7 × 10.sup.4                                           APUK (k,k)     9.2 × 10.sup.4                                           FPUK (q,q)    10.9 × 10.sup.4                                           ACUK (--,q)   15.7 × 10.sup.4                                           FCUK (--,q)   16.8 × 10.sup.4                                           V.sub.1 P (k,q)                                                                              9.7 × 10.sup.4                                           V.sub.2 P (k,q)                                                                             10.4 × 10.sup.4                                           ______________________________________                                    

FIGS. 5-1 and 5-2 collectively depict the nucleotide sequence encodingthe human prourokinase-like polypeptide FCUK (-,q), and thecorresponding amino acid sequence using single letter abbreviations.

FIGS. 6-1, 6-2 and 6-3 collectively depict the nucleotide sequenceencoding the human prourokinase-like polypeptide AHUK (q,q), and thecorresponding amino acid sequence using single letter abbreviations.

FIGS. 7-1, 7-2 and 7-3 collectively depict the nucleotide sequenceencoding the human prourokinase-like polypeptide APUK (k,k), and thecorresponding amino acid sequence using single letter abbreviations.

FIGS. 8-1, 8-2 and 8-3 collectively depict the nucleotide sequenceencoding the human prourokinase-like polypeptide APUK (k,q), and thecorresponding amino acid sequence using single letter abbreviations.

Furthermore, the living organism described herein have been deposited atthe Fermentation & Research Institute, 1-3, Higashi 1-Chome,Tsukaba-shi, Ibaraki-ken, 305, Japan.

Now that the invention has been described,

We claim:
 1. A substantially pure, enzymatically-active, humanprourokinase mutein comprising a human prourokinase derivative fusedwith an amino-terminal oligopeptide, said amino-terminal oligopeptidecapable of forming a covalent bond with fibrin present in a blood clotthrough the enzymatic action of human blood coagulation factor XIII,wherein said prourokinase derivative is derived from the amino acidsequence Ser¹ -Leu⁴¹¹ of human prourokinase and consists essentially ofan amino acid sequence selected from the group consisting ofLeu⁴-Leu⁴¹¹, Leu⁴ -Leu⁴¹¹ in which lysine¹³⁵ is substituted with a glutamineresidue, Leu⁴ -Leu⁴¹¹ in which lysine¹³⁵ and arginine¹⁵⁶ are bothsubstituted with glutamine residues, Leu⁴ -Leu⁴¹¹ in which lysine¹³⁵ issubstituted with an aspartate residue and arginine¹⁵⁶ is substitutedwith a glutamine residue, Leu¹⁴⁴ -Leu⁴¹¹, and Leu¹⁴⁴ -Leu⁴¹¹ in whicharginine¹⁵⁶ is substituted with a glutamine residue; and, wherein saidamino terminal oligopeptide is selected from the group consisting of(Met.)Asn.Gln.Glu.Gln.Val.Ser.Pro.Leu.Thr.Leu.Leu.Lys,(Met.)Gln.Ala.Gln.Gln.Met.Val.Gln.Pro.Gln.Ser.Pro.Val.Ala.Val.Lys,(Met.)Ala.Gln.Lys.Met.Val.Gln.Pro.Leu.Thr.Leu.Leu.Lys,(Met.)Ala.Gln.Lys.Met.Val.Gln.Pro.Gln.Thr.Leu.Leu.Lys,(Met.)Gln.Glu.Gln.Val.Ser.Pro.Gln.Thr.Leu.Leu.Lys,(Met.)Asn.Gln.Asp.Gln.Val.Ser.Pro.Gln.Thr.Leu.Leu.Lys,(Met.)Asn.Gln.Glu.Asn.Val.Ser.Pro.Gln.Thr.Leu.Leu.Lys, and,(Met.)Asn.Gln.Ser.Met.Val.Ser.Pro.Gln.Thr.Leu.Leu.Lys.
 2. An isolatedand purified DNA segment consisting essentially of a region encoding ahuman prourokinase mutein of claim 1 an allelic variant or exhibitingequivalent biological activity, capable of hybridizing therewith underconditions allowing detection of 70%.
 3. DNA segment of claim 2 furthercomprising a transcriptional promoter element in operable linkage withsaid region encoding a human prourokinase mutein, said promoter elementuseful as a promoter of gene expression in E. coli.
 4. A DNA constructcomprising a DNA sequence of claim 2 and a replication system capable ofreplication in an E. coli host cell.
 5. A recombinant plasmid comprisinga DNA sequence of claim 2 wherein said plasmid is a cloning vehicle andis capable of expressing a DNA sequence of claim 2 in a transformed E.coli host cell.
 6. E. coli transformed with the plasmid according toclaim
 5. 7. The E. coli transformants of the DNA construct of claim 4selected from the E. coli strain transformants consisting essentially ofFERM BP-2467, FERM BP-2465, and FERM BP-2466.